The pancreas is composed of two morphologically distinct compartments: exocrine cells, encompassing acinar and duct cells, and the endocrine tissue (reviewed in Slack, 1995). The endocrine compartment represents less than 2% of the organ and consists of five hormone-synthesising cell types: the insulin-secreting β-cells, the glucagon-secreting α-cells, the somatostatin-secreting δ-cells, pancreatic polypeptide-secreting PP-cells and the ghrelin-secreting ε-cells. These endocrine cells are organised into highly vascularised functional units called islets of Langerhans that are involved in glucose homeostasis. Pancreatic development corresponds to an elaborate process of morphological events accompanied by a complex pattern of cellular differentiation and lineage selection. These events are mediated in great part by tissue interactions, signalling pathways and directed cascades of gene expression that determine cell fate. Thus, Pdx1 plays a critical role in early pancreas development: removal of PDX1 by gene targeting arrests pancreatic development after initial bud formation. The specification of endocrine cells in the developing pancreatic endoderm depends on appropriate Notch signalling and expression of the pro-endocrine basic helix-loop-helix (bHLH) transcription factor neurogenin 3 (Ngn3), a member of the neurogenin/neuroD family of pro-neuronal genes. Endocrine cell fate allocation largely depends on the interplay between the transcription factors Arx and Pax4. These were shown to display antagonistic activities in the processes underlying the specification of the endocrine subtype destinies through an inhibitory cross-regulatory circuit that controls the transcriptional state of these two genes (Collombat et al., 2005). The expression of Arx was found to be upregulated in the absence of a functional Pax4 allele and vice versa (augmentation of the Pax4 transcript content in Arx mutants), suggesting that normal endocrine specification requires the prevalence of either factor in order to specify a particular endocrine cell subtype; if Pax4 predominates, the β- and δ-cell fates will be specified, whereas Arx will favour the α-cell commitment. Lastly, Arx and Pax4 were found to mutually inhibit each other's transcription through direct physical interaction with the pertinent promoter (Collombat et al., 2005). The persistent expression of Arx in early pancreatic and/or endocrine precursor cells during embryonic development resulted in a dramatic reduction in β- and δ-cell numbers, concurrent with an increase in α- and PP-cell populations (Collombat et al., 2007). These results indicated that Arx is thereby sufficient to promote the α- and PP-lineages during pancreas morphogenesis. More interestingly, the misexpression of Arx in adult β-cells was found to induce their conversion into cells exhibiting α- or PP-cell characteristics (Collombat et al., 2007). This discovery was of fundamental importance in the context of β-cell-based therapy, as it implied that the complementary conversion might be achieved, that is, to generate β-cells from alternative endocrine cells. Accordingly, the ectopic expression of Pax4 in the developing mouse pancreas resulted in oversized islets mostly composed of cells displaying a β-cell phenotype (Collombat et al., 2009), indicating that Pax4 misexpression is sufficient to promote the β-cell lineage allocation during the development. Importantly, the misexpression of Pax4 in glucagon-expressing cells, coupled with genetic labelling to follow their outcome, resulted in a loss of α-cells concurrently with a dramatic increase in the number of insulin-producing cells. Lineage tracing experiments demonstrated a conversion of glucagon-positive cells into insulin-expressing cells. Moreover, the newly generated insulin-expressing cells showed most of the features of true β-cells (Collombat et al., 2009).
Accordingly, international patent application WO 2011/012707 describes a method for producing a population of pancreatic beta-cells, comprising the step of providing at least one pancreatic alpha-cell or at least one precursor cell with Pax4 or a nucleic acid encoding Pax4. However, due to (1) the mutual inhibitory interplay occurring between Pax4 and Arx and (2) the expression of Arx in glucagon-producing cells, it remains to determine whether the ectopic expression of Pax4 in glucagon-expressing cells and the ensuing α-to-β-like cell conversion were directly caused by Pax4 or by the subsequent inhibition of Arx expression. Furthermore, it should be reminded that hitherto Arx and Pax4 have only been shown to mutually inhibit during embryogenesis and particularly during the genesis of the pancreas and never in adult pancreatic beta-cells (Kordowich et al., 2011).
The regenerative capacity of pancreatic alpha-cells (or glucagon-producing cells), and their potential for conversion into β-like cells by the simple ectopic expression of Pax4, are of interest in the context of T1D research. However, this transgenic approach would be unfeasible for the development of human-targeted therapies.
Thus, the possibility to produce β-cells from α-cells by directly inhibiting Arx in said cells (e.g. in adult β-cells) has not been experimentally demonstrated nor even suggested, whereas there is need for an easy and safe method that generates β-cells that can be used in the therapeutic field (in vivo) as well as in the screening field (in vitro). Additionally, identifying small compounds mimicking the effects of the inhibition of Arx and/or inducing the conversion of pancreatic alpha-cells in pancreatic beta-cells is highly required.
Over the past decades, numerous studies have uncovered a role for γ-aminobutyric acid (GABA) in the endocrine pancreas and in diabetes mellitus. GABA, synthesized from glutamate by glutamic acid decarboxylase (GAD) in β-cells, is an extracellular signaling molecule in the pancreatic islets. Once released, GABA is thought to serve as a functional regulator of pancreatic hormone secretion or as a fast-acting paracrine signaling molecule for the communication between β-cells and the other endocrine cells in the islets of Langerhans. The presence of GABAB receptors in β-cells and of GABAA receptors in α-cells supports the putative autocrine/paracrine role of locally secreted GABA in islets. Indeed, the GABA regulation of hormone secretion was shown to be mediated by its receptors. Interestingly, GAD (the enzyme involved in GABA synthesis) was found to be one of the major autoantigens in T1D and GABA was found to be decreased in endocrine pancreatic tissue in experimental and human diabetes. These findings clearly indicate that the GABA network is altered in T1D but its role in this pathology remains to be clarified. In addition, several studies have demonstrated that GABA participates in maintaining β-cell mass (Mendu et al., 2011; Soltani et al., 2011), inducing β-cell proliferation and protecting β-cells from apoptosis in vitro (Soltani et al., 2011; Ligon et al., 2007). Interestingly, it was shown that GABA could decrease blood glucose levels and exert protective and regenerative effects on the β-cell mass in streptozotocin-induced diabetes in mice (Soltani et al., 2011; Gomez et al., 2007). GABA was also found to reverse diabetes in NOD mice (Soltani et al., 2011; Tian et al., 2011). The suggested explanation was that GABA might act in an autocrine/paracrine manner to regenerate the pancreatic islets via β-cell proliferation.